An Agonist Dependent Allosteric Antagonist of Prostaglandin EP2 Receptors

All reported prostaglandin EP2 receptor antagonists have a purely orthosteric, competitive mode of action. Herein, we report the characterization of compound 1 (pubchem CID 664888) as the first EP2 antagonist that features a reversible, agonist dependent allosteric mode of action. Compound 1 displayed an unsurmountable inhibition of cAMP accumulation stimulated by different EP2 agonists in C6 glioma cells overexpressing human EP2 (C6G-hEP2). The degree of reduction of agonist potency and efficacy depended on the agonist employed. Negative allosteric modulation was not observed in C6G cells overexpressing human EP4, IP, or DP1 receptors. Moreover, in the murine microglial cell line that stably expresses human EP2 receptors (BV2-hEP2), compound 1 reduced the EP2 agonist-induced elevation of interleukin 6 (IL-6), IL-1β, and hEP2 mRNA levels and increased that of tumor necrosis factor (TNF)-α. Compound 1 was docked into a homology model of hEP2. The predicted binding site on the cytoplasmic receptor surface was similar to that of allosteric inhibitors of the β2-adrenergic, CC chemokine receptor 9 (CCR9), and CC chemokine receptor 2 (CCR2) receptors, which supports the notion of a conserved G-protein-coupled receptor (GPCR) binding pocket for allosteric inhibitors. As the first agonist dependent negative allosteric modulator of EP2 receptor, the structure of this compound may provide a basis for developing improved allosteric modulators of EP2 receptors.